Transneuronal labeling of a nociceptive pathway, the spino-(trigemino-)parabrachio-amygdaloid, in the rat

Abstract

Transneuronal tracing of a nociceptive pathway, the spino-(trigemino)-parabrachio-amygdaloid pathway, was performed using an alpha-herpes virus, the Bartha strain of pseudorabies virus (PRV). Microinjection of PRV into the central nucleus of the amygdala (Ce) resulted in progressive retrograde and transneuronal infection of a multisynaptic circuit involving neurons in the brainstem and spinal cord as detected immunocytochemically. At short survival (26 hr), retrogradely labeled neurons were concentrated in the external lateral nucleus of the parabrachial complex (elPB) but were absent from both the trigeminal nucleus caudalis (TNC) and the spinal cord. At longer survivals (52 hr), labeled cells were present in lamina I of both the TNC and spinal dorsal horn. Retrograde labeling from the Ce with Fluoro-gold demonstrated that elPB neurons have long dendrites extending laterally into the terminal field of spinal and trigeminal afferents, where transneuronal passage of PRV to these afferents could occur. Even longer survivals (76 hr) resulted in a columnar pattern of cell labeling in the TNC and spinal dorsal horn that extended from lamina I into lamina II. At this longest survival, primary sensory neurons became infected. Bilateral excitotoxic lesions of the elPB blocked almost all viral passage from the Ce to superficial laminae of the TNC and spinal dorsal horn. These results demonstrate that nociceptive input to the amygdala is relayed from neurons in lamina I through the elPB. We propose that this modular arrangement of lamina I and II neurons may provide the basis for spinal processing of peripheral input to the amygdala.

Fig. 4.

Cases 47, 40, 67, 71, 93. Diagram of PRV and Fluoro-gold injection sites in the amygdala plotted onto section reproduced from the atlas of Swanson (1992). The location of the injection sites was determined from the distribution of gold particles and from the cannula tracts.

Fig. 1.

Case 97. Bilateral PRV microinjections into the lateral thalamus (arrows). A, The injection on the left was made 22 min and the one on the right 59 min before perfusion. Note that the immunoreactivity on theleft is well localized, implying a restricted diffusion of the virus. By contrast, little immunoreactivity is present when the survival time was ∼1 hr. B, The injection on theleft side above was imaged at high power, to show the dust-like, noncellular appearance of the immunolabeling. Third ventricle (III); internal capsule (ic). Scale bars: A, 400 μm;B, 33 μm.

Fig. 2.

A, Case 47. After 26 hr, PRV-immunoreactive neurons are visible at the injection site (arrow) in cell bodies. The inset shows PRV immunolabeled cell profiles (arrowheads) and colloidal gold (arrow). Compare with the dust-like immunoreactivity in Figure 1B. B, Case 93. An example of a PRV injection site in the Ce (lateral division) at 52 hr. The injection site is localized by gold particles, which persist after coinjection with the virus. PRV immunoreactivity is no longer found at the injection site as inA, which is likely attributable to clearance of the virus by an immune response (Rinaman et al., 1993; Card and Enquist, 1995). Labeled cells are visible in the perirhinal (PR) and piriform cortex (Pir); these presumably resulted from both direct and transneuronal transport. Basolateral amygdaloid nucleus (BLA); central nucleus (Ce); caudate putamen (CP); external capsule (ec). Scale bars: A(inset), 33 μm; A, 250 μm;B, 150 μm.

Fig. 3.

A, Case 92. PRV-immunoreactive neurons at the injection site in the lateral thalamus; the center of the injection site is 520 μm from the medial border of the ventroposterolateral nucleus (VPL). The survival time was 72 hr. B, Case 89. PRV-immunoreactive neurons at the injection site in the lateral thalamus; the center of the injection site is 320 μm from the medial border of the VPL nucleus. The survival time was 77 hr. Reticular nucleus of the thalamus (Rt); ventroposteromedial nucleus (VPM). C, Case 92. Retrogradely labeled PRV neurons in the caudal medulla, at the same level as in Figure 3C, are present in the medial trigeminal nucleus caudalis (TNC) only. D, Case 89. Retrogradely labeled PRV-immunoreactive neurons in the caudal medulla are present in both the gracile nucleus (Gr;arrowhead) and in the medial part of the trigeminal nucleus caudalis (TNC; arrow). Cuneate nucleus (Cu). Scale bar: 300 μm.

Fig. 5.

PRV immunoreactivity in the ipsilateral parabrachial area and caudal medulla after PRV injection in the Ce. Case 47. A, Parabrachial area 26 hr after injection in the Ce (see Fig. 4). The retrogradely labeled neurons were largely restricted to the elPB. B, There is no labeling in the trigeminal nucleus caudalis (TNC) 26 hr after PRV injection in the Ce. Case 40. C, PRV immunoreactivity in the ipsilateral parabrachial area 46 hr after injection in the Ce. Cusp-shaped islets formed of PRV-labeled cell bodies (arrowheads) extend into the lateral crescent area from the el PB. D, With this survival time, we found very few PRV-immunoreactive neurons in lamina I of the ipsilateral caudal medulla (arrow). Case 93. E, PRV immunoreactivity in the ipsilateral parabrachial area 52 hr after injection in the Ce. F, PRV-immunoreactive neurons were observed at somewhat regular intervals in lamina I of the trigeminal nucleus caudalis (arrows). Occasionally we found two adjacent cells that spanned the dorsoventral extent of lamina I. Case 71. G, PRV immunoreactivity in the ipsilateral parabrachial area 76 hr after injection in the Ce. H, In the ipsilateral caudal medulla, we recorded clusters of PRV-immunopositive neurons that included neurons in lamina I and in the ventrally subjacent lamina II. As for F, the clusters were located at somewhat regular intervals. External lateral nucleus (elPB); Kölliker–Fuse (KF); lateral crescent area (lc); medial nucleus (M); superior cerebellar peduncle (scp). Scale bar: 150 μm.

Fig. 6.

Case 71. Characteristics of injection site after coinjection of PRV and Fluoro-gold. Immunostaining for PRV (A) shows the cannula tract and a small area of tissue necrosis. The center of injection is at the junction of the lateral Ce and the basolateral nucleus (BLA). Note that the injection site is almost devoid of PRV immunoreactivity. The perirhinal (PR) and piriform cortex (Pir), however, contain retrogradely labeled neurons. An adjacent section immunostained for Fluoro-gold (B) demonstrates the much greater radius of diffusion and the consequent more extensive retrograde labeling of neurons that project to the amygdala (see Table 2). Optic tract (opt). Scale bar: 500 μm.

Fig. 7.

Case 71. PRV immunoreactive neurons in the trigeminal ganglion ipsilateral to the Ce injection. Labeled cells (arrows) demonstrate a dark reaction product; most of these are of small diameter. By contrast, unlabeled cells (arrowheads), visible because of light background staining, are larger. Scale bar: 100 μm.

Fig. 8.

A, Case 71. High magnification of trigeminal ganglion on the side of PRV injection in the Ce. The tissue has been double labeled for PRV (red immunofluorescence) and the neurotransmitter substance P (yellow immunofluorescence). Two cells labeled exclusively for substance P are evident (single arrowhead), as well as one for PRV only (arrow). One example of a double-labeled cell is indicated by a double arrowhead. The light red cytoplasmic fluorescence of many large cells is background signal only. B, Case 101. Trigeminal nucleus caudalis double labeled for PRV injected in the Ce (brown reaction product) and cholera toxin injected in the elPB (black reaction product). Cholera-toxin-labeled neurons are present mostly in lamina I, with a few in laminae III, IV, and V. PRV labeling is confluent in lamina I and II consistent with the longer time post-injection (96 hr). All cholera toxin-labeled cells were double labeled for PRV, similar to case 51, for which the two tracers were injected in the parabrachial (Fig. 12). More importantly, the dendritic profiles of cholera- toxin-labeled lamina I parabrachial projection cells are seen to extend into lamina II (arrowheads). Scale bars:A, 50 μm; B, 100 μm.

Fig. 9.

Case 1501. A, B, Low- and high-power views of a Nissl-stained section through an ibotenic acid lesion in the lateral parabrachial. Disruption of the cellular architecture in the elPB and vicinity (boxed area inA) is evident at high magnification (B).Arrowheads delimit the lesioned area in which normal appearing perikarya are no longer recognized. The arrowpoints to a cluster of pyknotic perikarya within the lesion. Theinterrupted line delineates the superior cerebellar peduncle (scp). C, D, PRV immunoreactivity. C, In the pons, the parabrachial area is filled with immunopositive neurons, with the exception of the region of the elPB (arrows). In the caudal medulla, abundant labeling is observed in the nucleus of the solitary tract (NTS), whereas moderate labeling is present in the reticular formation (Ret). Several immunopositive cells (arrowheads) are present in lamina I of the trigeminal nucleus caudalis (TNC). Scale bars: A,C, D, 500 μm; B, 200 μm

Fig. 10.

Case 67. Retrograde labeling with Fluoro-gold in the parabrachial complex after injection in the Ce (for injection site, see Fig. 4). Dense labeling is present in the elPB (not labeled for clarity); isolated cell bodies are present in the medial (M) and ventral lateral (vl) subnuclei. Two striking features characterize the pattern of labeling in the elPB. First, as we found after retrograde PRV labeling from the amygdala, cusp-shaped islets formed of cell bodies (arrowheads) extend into the lateral crescent area (lc) from the elPB. Second, long dendrites extend from the elPB neurons and islets into the lc, the Kölliker–Fuse (KF), and the superior lateral subnucleus (sl). Scale bar: 120 μm.

Fig. 11.

Case 103. A, PRV coinjected with gold particles (arrowheads) into theelPB and lateral crescent area (lc) nuclei of the parabrachial complex. B, PRV immunolabeling in the caudal medulla 56 hr after injection. We recorded clusters of labeled neurons (arrowheads) that spanned laminae I throughout the extent of the trigeminal nucleus caudalis (TNC) and occasionally encroached on lamina II. Although we found no labeled cells in the deeper laminae of the TNC, some were found in the reticular formation (Rt). PRV immunolabeling in the cervical spinal cord (C) revealed neuronal clusters in laminae I and II (arrowheads).D, Retrograde PRV labeling was also found in the central nucleus of the amygdala (Ce), the amygdalostriatal junction (AStr), and the lateral hypothalamus (LH) after this elPB injection. Basolateral amygdaloid nucleus, anterior (BLA); dorsal root (DR); dorsal funiculus (DF); lateral funiculus (LF); optic tract (opt); reticulated area of the spinal dorsal horn (Ret). Scale bars: A, B,D, 250 μm; C, 125 μm.

Fig. 12.

Case 51. A, PRV immunolabeling in the caudal medulla 96 hr after coinjection of PRV and CTB in the elPB. Densely packed PRV-immunopositive neurons were found throughout the extent of laminae I and II of the trigeminal nucleus caudalis (TNC). The abundance of labeling obscured the columnar organization. B, Double labeling for cholera toxin B and PRV demonstrated cholera toxin B-immunoreactive neurons; these were largely restricted to lamina I (arrows) and were always immunoreactive for PRV. PRV-immunoreactive neurons (arrowheads) were seen to underlie double-labeled lamina I neurons (arrows). Compared with Figure8B, the cholera toxin-labeled lamina I neurons appear atrophic and their dendritic tree is not evident. This is likely attributable to a longer (∼24 hr) presence of viral replication in these cells. C, PRV immunolabeling in the cervical spinal cord demonstrated elongated, radially oriented neuronal columns that spanned laminae I and II; the columns are spaced at regular intervals (130–360 μm). Isolated PRV cells are also seen in the reticulated part of the dorsal horn (Ret), the area surrounding the central canal (cc), and in lamina VII of the ventral horn. D, At higher magnification, it is apparent that the columnar immunolabeling is restricted to laminae I and II, and that some bridging of columns has begun to occur in lamina I such that at later survivals, the columns could be obscured. Scale bars: A, 300 μm; B, 175 μm;C, 50 μm; D, 100 μm.